The chief goal is to understand how the hormone vasopressin regulates water excretion by the kidney. Vasopressin's action is mediated through regulation of the molecular water channel aquaporin-2. Based on our studies a decade ago, it is now clear that vasopressin regulates aquaporin-2 in a time frame of seconds to minutes by altering the distribution of the water channel aquaporin-2 between the plasma membrane and the cytoplasm via vesicular trafficking. Trafficking of aquaporin-2 to the plasma membrane renders the cells permeable to water. We are presently using a systems approach to address the mechanisms involved. For this approach, we are integrating protein mass spectrometry, DNA microarrays, mathematical modeling and physiological methods. The following is a summary of work over the past year appearing in the 12 references published from August, 2009 until August, 2010. The first 5 references in the reference list below show publications that have used protein mass spectrometry (1-5) to investigate protein networks involved in regulation of renal water and solute transport, as well as the mass spectrometry tools developed for these studies. The next 2 references (6,7) deal with studies of vasopressin action in regulation of aquaporin-2 and urea transporters using conventional physiological and immunochemical techniques. The next 2 (8,9) are clinically-oriented papers which describe bioengineering work to exploit our recent discovery that normal kidneys excrete exosomes in the urine. Exosomes are small membrane particles secreted by every cell type facing the urinary space in the kidney. The goal of these studies is to develop the methods infrastructure to allow clinical investigators to isolate urinary exosomes for disease biomarker studies. The next 2 references describe work focusing on the use of animal models of disease processes to discover the pathophysiological basis of salt and water imbalance disorders (10, 11). These studies address mechanism and treatment of water balance disorders associated with congestive heart failure and X-linked nephrogenic diabetes insipidus. The 12th paper is a commentary article in the journal Nature. References 1. Gunaratne R, Braucht DW, Rinschen MM, Chou CL, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals cAMP/vasopressin-dependent signaling pathways in native renal thick ascending limb cells. Proc Natl Acad Sci U S A. 2010 Aug 16. Epub ahead of print PubMed PMID: 20713729. 2. Da Silva N, Pisitkun T, Belleanne C, Miller LR, Nelson R, Knepper MA, Brown D, Breton S. Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting. Am J Physiol Cell Physiol. 2010 Jun;298(6):C1326-42. Epub 2010 Feb 24. PubMed PMID: 20181927;PubMed Central PMCID: PMC2889637. 3. Rinschen MM, Yu MJ, Wang G, Boja ES, Hoffert JD, Pisitkun T, Knepper MA. Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor-dependent signaling pathways in renal collecting duct cells. Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3882-7. Epub 2010 Feb 5. PubMed PMID: 20139300;PubMed Central PMCID: PMC2840509. 4. Bansal AD, Hoffert JD, Pisitkun T, Hwang S, Chou CL, Boja ES, Wang G, Knepper MA. Phosphoproteomic profiling reveals vasopressin-regulated phosphorylation sites in collecting duct. J Am Soc Nephrol. 2010 Feb;21(2):303-15. Epub 2010 Jan 14. PubMed PMID: 20075062;PubMed Central PMCID: PMC2834543. 5. Tchapyjnikov D, Li Y, Pisitkun T, Hoffert JD, Yu MJ, Knepper MA. Proteomic profiling of nuclei from native renal inner medullary collecting duct cells using LC-MS/MS. Physiol Genomics. 2010 Feb 4;40(3):167-83. Epub 2009 Dec 8. PubMed PMID: 19996160;PubMed Central PMCID: PMC2825761. 6. Hwang S, Gunaratne R, Rinschen MM, Yu MJ, Pisitkun T, Hoffert JD, Fenton RA, Knepper MA, Chou CL. Vasopressin Increases Phosphorylation of Ser84 and Ser486 in Slc14a2 Collecting Duct Urea Transporters. Am J Physiol Renal Physiol. 2010 Jun 24. Epub ahead of print PubMed PMID: 20576681. 7. Xie L, Hoffert JD, Chou CL, Yu MJ, Pisitkun T, Knepper MA, Fenton RA. Quantitative analysis of aquaporin-2 phosphorylation. Am J Physiol Renal Physiol. 2010 Apr;298(4):F1018-23. Epub 2010 Jan 20. PubMed PMID: 20089674;PubMed Central PMCID: PMC2853310. 8. Gonzales PA, Zhou H, Pisitkun T, Wang NS, Star RA, Knepper MA, Yuen PS. Isolation and purification of exosomes in urine. Methods Mol Biol. 2010;641:89-99. PubMed PMID: 20407943. 9. Fernndez-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int. 2010 Apr;77(8):736-42. Epub 2010 Feb 3. PubMed PMID: 20130532. 10. Li JH, Chou CL, Li B, Gavrilova O, Eisner C, Schnermann J, Anderson SA, Deng CX, Knepper MA, Wess J. A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic diabetes insipidus. J Clin Invest. 2009 Oct;119(10):3115-26. PubMed PMID: 19729836;PubMed Central PMCID: PMC2752083. 11. Ltken SC, Kim SW, Jonassen T, Marples D, Knepper MA, Kwon TH, Frkiaer J, Nielsen S. Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II AT1 receptor blockade. Am J Physiol Renal Physiol. 2009 Dec;297(6):F1678-88. Epub 2009 Sep 23. PubMed PMID: 19776175;PubMed Central PMCID: PMC2801339. 12. Knepper MA, Mindell JA. Structural biology: Molecular coin slots for urea. Nature. 2009 Dec 10;462(7274):733-4. PubMed PMID: 20010678.